ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2011, p. 1237–1247
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 3
Comparative Studies Evaluating Mouse Models Used for
Efficacy Testing of Experimental Drugs against
Mary A. De Groote,1Janet C. Gilliland,1Colby L. Wells,1Elizabeth J. Brooks,1Lisa K. Woolhiser,1
Veronica Gruppo,1Charles A. Peloquin,2Ian M. Orme,1and Anne J. Lenaerts1*
Mycobacterial Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins,
Colorado 80523,1and Infectious Diseases Pharmacokinetics Laboratory, University of Florida, Gainesville, Florida 326102
Received 29 April 2010/Returned for modification 5 July 2010/Accepted 29 November 2010
Methodologies for preclinical animal model testing of drugs against Mycobacterium tuberculosis vary from
laboratory to laboratory; however, it is unknown if these variations result in different outcomes. Thus, a series
of head-to-head comparisons of drug regimens in three commonly used mouse models (intravenous, a low-dose
aerosol, and a high-dose aerosol infection model) and in two strains of mice are reported here. Treatment with
standard tuberculosis (TB) drugs resulted in similar efficacies in two mouse species after a low-dose aerosol
infection. When comparing the three different infection models, the efficacies in mice of rifampin and pyrazin-
amide were similar when administered with either isoniazid or moxifloxacin. Relapse studies revealed that the
standard drug regimen showed a significantly higher relapse rate than the moxifloxacin-containing regimen.
In fact, 4 months of the moxifloxacin-containing combination regimen showed similar relapse rates as 6
months of the standard regimen. The intravenous model showed slower bactericidal killing kinetics with the
combination regimens tested and a higher relapse of infection than either aerosol infection models. All three
models showed similar outcomes for in vivo efficacy and relapse of infection for the drug combinations tested,
regardless of the mouse infection model used. Efficacy data for the drug combinations used also showed similar
results, regardless of the formulation used for rifampin or timing of the drugs administered in combination.
In all three infection models, the dual combination of rifampin and pyrazinamide was less sterilizing than the
standard three-drug regimen, and therefore the results do not support the previously reported antagonism
between standard TB agents.
For the first time in many years, there is a portfolio of
promising new compounds at every level of tuberculosis (TB)
drug discovery and development (4; www.tballiance.org). How-
ever, careful selection of new drug candidates is imperative,
and efficient screening models for new drugs, including perti-
nent animal models, need to be further developed and studied.
Preclinical testing in animals of newly discovered agents alone,
and in combination with new and old agents, prior to being
tested in humans is a crucial but lengthy process. These drug
regimens include agents that provide bactericidal activities
against rapidly growing bacilli, but they especially aim to in-
clude those that possess potent sterilizing activities and hence
prevent relapse (8). The most commonly used animal species
in TB drug development is the mouse, mainly because of eco-
nomical and practical reasons, but also because of the limited
requirement of compound (26).
Various mouse Mycobacterium tuberculosis infection models
are utilized by both industry and academia, and they differ in
the route of infection with M. tuberculosis, inoculum and strain
of M. tuberculosis, strain of mice, timing of the start of treat-
ment after infection, the length of treatment, etc. A model
where therapy is started immediately after infection is ideally
suited in order to quickly assess whether a compound has
sufficient bioavailability, adequate absorption after oral admin-
istration, and metabolism sufficient to result in in vivo efficacy.
A more realistic model might be one in which there is time for
the disease to establish and produce granulomas, i.e., more
consistent with what is seen in human tuberculosis. Models can
also vary based on inoculum size, and both high- and low-dose
inocula of M. tuberculosis were included in our study. We
define these infection models based on inoculum size as fol-
lows. In the “low-inoculum” infection mouse model, mice de-
velop an adequate immune response and can survive for
months to more than a year after infection (22), whereas in the
“high-inoculum” infection model, the immune response gets
overwhelmed quickly and the animals succumb to disease
within weeks after infection. There are various infection pro-
tocols based on the route of infection in mice. In the low-dose
aerosol (LDA) infection method, reported studies generally
aim at expected implantations in lungs of 30 to 100 CFU per
mouse (5, 6, 53), versus 3,000 to 10,000 CFU for high-dose
aerosol (HDA) infections (37, 39–41, 45, 47, 51, 52). The LDA
method aims for an infection with few bacilli, leading to a
chronic infection. The HDA method leads rapidly to progres-
sive disease, with bacterial numbers reflecting those of a hu-
man cavity (41), and the animals succumb to disease without
drug intervention. The route using an intravenous (i.v.) injec-
tion in the tail vein typically delivers a high-dose inoculum of
M. tuberculosis (21, 35, 48–50). Other mouse infection models
for TB drug evaluations have used the intratracheal (46) or
* Corresponding author. Mailing address: Department of Micro-
biology, Immunology and Pathology, Colorado State University,
Fort Collins, CO 80523. Phone: (970) 491-3079. Fax: (970) 491-
1815. E-mail: email@example.com.
?Published ahead of print on 6 December 2010.
intranasal (7) route of infection, which are used less frequently.
The route and site of inoculum may be critical, as the immune
response, especially the memory immune response and hence
the rate of relapse, may well depend on the route of delivery of
the pathogen. Mouse strains most commonly used for TB drug
evaluations are Swiss, BALB/c, and C57BL/6 mice (20, 28, 40,
41). Laboratories generally use their in-house-developed
mouse model, which is in most cases thoroughly validated
before routine preclinical evaluation of novel compounds.
However, to our knowledge, an effort to reevaluate side by side
the different mouse models used for TB drug development has
never taken place. In this comparative study, we interrogated
the LDA and HDA and also the i.v. infection models, based
on the rationale that these are the most widely used mouse
In this study, we administered different combinations of TB
drugs to mice and used these drug treatment regimens to
elucidate the most important variables within the different
tuberculosis mouse infection models. In addition, the goal was
to assess whether the outcome for in vivo efficacy and relapse
of infection for the drug combinations tested was the same or
different depending on the mouse infection model used. The
standard drug regimen of isoniazid (INH), rifampin (RIF), and
pyrazinamide (PZA) administered for up to 6 months was
evaluated. In addition, moxifloxacin (MXF) was also included
as a replacement for INH in the standard regimen; moxifloxa-
cin is a fluoroquinolone currently in phase 3 clinical trials for
TB treatment (1). The rationale for the inclusion of MXF-
containing combination regimens in this study was to evaluate
two relevant drug combination regimens for tuberculosis in the
three mouse infection models. In addition, the inclusion of
MXF-containing regimens in particular was aimed to see
whether it showed improved in vivo efficacy versus the standard
drug regimen, as seen in an earlier published mouse study (40),
and these mouse data formed the basis of a TB Trials Consor-
tium study (11). Our study is the first to directly compare the
various mouse models used in TB drug screening within the
same laboratory to identify the most critical parameters and
details in the methodology that may influence subsequent re-
MATERIALS AND METHODS
Mice. Female BALB/c or C57BL/6 mice (Charles River Laboratories, Wil-
mington, MA) between the ages of 6 and 12 weeks were housed at five animals
per cage in HEPA-filtered racks (Thoren Caging Systems Inc., Hazleton, PA) in
certified animal biosafety level three (ABSL-3) laboratories and were rested for
1 to 3 weeks before infection with M. tuberculosis.
Bacterial strains. The virulent M. tuberculosis strain Erdman (TMC 107;
ATCC 35801) was used as the standard strain for drug testing in all animal
studies (53). Drug susceptibility testing of the M. tuberculosis Erdman strain used
was recently performed for PZA and moxifloxacin at the Mycobacteriology
Laboratory at the National Jewish Health, Denver, CO, by Bactec (17, 18). MIC
testing for isoniazid and rifampin was performed by the microdilution plate
method (16). The pncA gene was sequenced by the U.S. Centers for Disease
Control and Prevention (Atlanta, GA). MICs were ?100 ?g/ml for pyrazin-
amide, 0.0078 ?g/ml for isoniazid, 0.0156 ?g/ml for rifampin, and ?2 ?g/ml for
moxifloxacin. No mutations in the pncA gene were detected.
Bacteria were originally grown as pellicles to generate seed lots (28). The
working stocks were generated by growing cultures to mid-log phase in
Proskauer-Beck medium containing 0.05% Tween 80 (Sigma Chemical Co., St.
Louis, MO), enumerated by colony counting on 7H11 agar plates, divided into
1.5-ml aliquots, and stored at ?70°C until use.
Antimicrobial agents and formulations. INH, PZA, and RIF were purchased
from Sigma Chemical Co. (St. Louis, MO). MXF was a generous gift of Bayer
Corporation in collaboration with the Global Alliance for TB Drug Develop-
ment. MXF was administered at 100 mg/kg of body weight, RIF at 10 mg/kg,
PZA at 150 mg/kg, and INH at 25 mg/kg. All antimicrobial compounds were
administered by oral gavage 5 days per week (at 0.2 ml per mouse). All drugs
were prepared in water except when specified differently.
Two methods of drug preparation were used for RIF, and two dosing sched-
ules of RIF were used in relation to the drugs in combination. The first method
consisted of the following conditions: RIF was ground in a clean mortar and
pestle to a small particle size prior to adding sterile distilled water (1, 39, 44).
INH, PZA, and MXF were dissolved in sterile distilled water as well. RIF was
dosed in the morning, and the other drugs were administered as combinations in
the afternoon (approximately 4 h apart). Drugs were prepared on the Friday
prior to the dosing week and kept at 4°C. A day before or on the day of dosing,
the drug combinations were combined. PZA solutions were incubated at 55°C on
the day of dosing until particles were dissolved.
The second method of RIF formulation and administration was the following:
RIF was dissolved in 100% dimethyl sulfoxide (DMSO) and slowly (dropwise)
diluted to a final concentration of 5% DMSO in water (28). INH, PZA, and
MXF were dissolved in autoclaved tap or deionized water. RIF was dosed at least
1 h after other drugs (but not more than 2 h). Concentrations of drugs were
adjusted monthly based on body weights. For combinations of INH and PZA,
concentrated stocks of individual drugs were prepared up to 7 days in advance,
combined the day before or the day of dosing, and left at room temperature (RIF
was kept at 4°C). PZA solutions, if particulate, were sonicated. In both methods,
all drugs were visibly completely dissolved and free of obvious particles at the
time of drug administration to the animal.
Infection models. Six- to 12-week-old BALB/c or C57BL/6 mice were exposed
to an LDA, HDA, or an i.v. infection with the virulent M. tuberculosis Erdman in
ABSL-3 laboratories. All studies were approved after institutional review by the
Animal Care and Use Committee and the Biosafety Committee at Colorado
State University. All aerosol infections utilized an inhalation exposure system
(Glas-Col, Inc., Terre Haute, IN), as described before (27). In the LDA infection
experiment, 8- to 10-week-old female BALB/c and C57BL/6 mice were exposed
to M. tuberculosis Erdman, derived from a frozen bacterial stock, with a titer of
4.87 ? 107CFU per ml (27). A 5-ml inoculum containing 2 ? 106CFU per ml
in autoclaved deionized water was placed in the Glas-Col nebulizer with settings
of 10 compressed air and 50 main (negative air) standard cubic feet per hour
For the HDA model (40, 41), 8- to 10-week-old female BALB/c mice were
infected with a freshly grown culture of M. tuberculosis. Bacteria were grown in
7H9 medium (Difco Inc., Lawrence, KS) supplemented with 10% oleic acid–
albumin–dextrose–catalase and Tween 80 and propagated to late log phase with
an optical density at 600 nm (OD600) of 0.8 to 1.0. Ten milliliters of the freshly
grown M. tuberculosis culture was placed in the Glas-Col nebulizer with settings
of 13 to 17 SCFH compressed air and 80 SCFH main (negative air). The
procedures included a 15-min preheat cycle, a nebulizing cycle of 30 to 40 min,
a cloud decay cycle of 15 to 30 min, with decontamination for 15 min (Glas-Col,
For i.v. infections, 8- to 12-week-old mice were injected with M. tuberculosis
Erdman with 6 to 7 log10CFU delivered in 0.1 ml of sterile phosphate-buffered
saline via injection of the lateral tail vein. Bacteria were suspended repetitively
through a SurGuard safety hypodermic needle (26 gauge; VWR, Wilmington,
DE) in order to obtain a single-cell bacterial suspension. Enumeration of the M.
tuberculosis inoculum from all infection routes was determined by CFU counts
on 7H11 agar plates (as described below). The actual bacterial load delivered to
the animals was determined from three mice per group the day after the infection
in the lungs from all aerogenically challenged animals and in lungs and spleens
from five mice in the i.v.-infected group. At the start of treatment (defined by
convention as day zero), the bacterial load was determined in lungs and spleens.
The timing of the drug treatment varied depending on the infection model being
evaluated and was based on published and unpublished data. Treatment regi-
mens ranged from 1 to 6 months with 5 to 8 mice per treatment group for each
sacrifice point during treatment and 10 to 22 mice per group for assessment of
relapse of infection. To assess relapse, animals were observed without drug
intervention for 3 months.
Enumeration of bacteria from tissues. After completion of therapy, mice were
sacrificed by CO2inhalation. After euthanasia, lungs, spleens, and/or livers were
aseptically removed and homogenized in Pyrex tubes containing 1 ml (after more
than 1 month of treatment) or 4.5 ml (when less than a month had elapsed since
start of therapy) of sterile saline. The number of viable organisms was deter-
mined by serial dilution in sterile physiologic saline of the homogenates plated on
1238DE GROOTE ET AL.ANTIMICROB. AGENTS CHEMOTHER.
nutrient 7H11 agar plates containing glycerol, oleic acid, dextrose, catalase,
albumin, and cycloheximide (GIBCO-BRL, Gaithersburg, MD). Plates were
incubated at 37°C in ambient air for 4 to 8 weeks prior to counting CFU. For
determination of the emergence of RIF-resistant colonies, tissue homogenates
were plated on 7H11 plates containing 4 ?g/ml of RIF. RIF resistance plating
was performed when indicated on residual frozen organ homogenates plated
directly on RIF-containing plates.
Pharmacokinetic analysis. Uninfected 8-week-old BALB/c mice were admin-
istered a single oral dose by gavage of different RIF formulations (see “Antimi-
crobial agents and formulations,” above). For both formulation methods, whole
blood was obtained from mice by cardiocentesis immediately after CO2eutha-
nasia at 0, 0.5, 1, 2, 4, 8, and 14 h from three mice at each time point (times are
in reference to rifampin administration); whole blood was collected in hepa-
rinized syringes containing 4.3 USP units of heparin per syringe (Sigma Chemical
Co., St. Louis, MO), transferred into plasma separator tubes (BD microtainer;
BD Diagnostics, VWR Wilmington, DE), and centrifuged at 4°C (5,410 rpm for
15 min). Plasma samples were snap-frozen in cryovials in liquid nitrogen and
stored at ?70°C prior to analysis at the Infectious Disease Pharmacokinetics
Laboratory (University of Florida, Gainesville, FL), where the samples were
analyzed by validated high-pressure liquid chromatography (RIF and INH) or
gas chromatography/mass spectrometry (PZA) assays (1). The data on concen-
trations in plasma were analyzed by using WinNonlin (version 5.2.1, 2008; Phar-
sight, Mountain View, CA) with standard noncompartmental techniques in order
to determine the maximum concentration (Cmax), time of Cmax(Tmax), area
under the concentration-time curve (AUC0–?), half-life, volume of distribution,
Statistical analysis. The CFU counts were converted to log values (log10
CFU), which were then evaluated by analysis of variance (ANOVA) when log10
CFU values appeared approximately normally distributed and by the Wilcoxon
rank sum test when the log counts were predominantly ?1 log and contained a
large proportion of 0 values. BALB/c and C57BL/6 aerosol and BALB/c i.v.
routes were compared using one-way ANOVA. When the ANOVA F-test was
significant (P ? 0.05), pairs of groups were compared using two-sided contrasts
of means. Similarly, INH- and MXF-containing combination therapies were
compared using one-way ANOVA separately for each infection model (HDA,
LDA, and i.v.) and each time point, followed by pairwise contrasts of treatment
In the experiment that compared two formulations of four dosing schedule
treatments, log10CFU results were analyzed using a two-way (formulation by
treatment) ANOVA. If the interaction was significant (P ? 0.05), it was con-
cluded that the differences between treatment means depend on formulation,
and treatments were compared by pairwise contrasts, separately for each formu-
lation. If the interaction was not significant, the treatments were compared
averaging over formulation, and the formulations were compared averaging over
treatment. For comparison of relapse rates, Fisher’s exact test was used. Treat-
ments were compared within models, and nonsignificant treatments were com-
bined for comparisons of models.
Analysis was done separately for relapse rates in lungs and spleens for the
HDA and i.v. routes for the three regimens by using Fisher’s exact test. In
addition, the relapse rates for the different drug regimens were analyzed via
pairwise two-sided comparisons for every infection model individually, again with
Fisher’s exact test to identify the regimens with similar and improved relapse
BALB/c and C57BL/6 mice after aerosol infection respond
similarly to treatment with standard drugs, whereas mice in-
fected via the i.v. route show a delayed treatment effect. The
purpose of this experiment was to evaluate the treatment ef-
ficacy of the standard drug regimen (INH, RIF, and PZA)
given over 4 months in two mouse strains (BALB/c and
C57BL/6) infected by LDA in one single experiment. In addi-
tion, parallel groups were also infected by i.v. injection in
BALB/c mice only (Table 1). After 1 month of treatment, the
bacterial loads were significantly different between the three
different infection models (P ? 0.0001, ANOVA F-test) (Fig.
1A), with the bacterial burden in lungs of the i.v.-infected
BALB/c mice higher than in the aerosol-infected BALB/c and
C57BL/6 mice (P ? 0.0001 for both mouse strain). The bacte-
rial loads in the lungs of both mouse strains infected by aerosol
were similar after 1 month of treatment (P ? 0.25) (Fig. 1A).
After 2 months of treatment the lung bacterial loads showed
similar results, with the i.v.-infected mice showing higher bac-
terial loads again than the aerosol-infected BALB/c and
C57BL/6 mice (P ? 0.020 and 0.019, respectively). The treat-
ment efficacies in the two mouse strains infected by aerosol
were again statistically similar (P ? 0.9) (Fig. 1A). After 4
months, results in the lungs showed bacterial numbers were
reduced to only a few bacilli, and this finding was in a single
mouse. These results could not be compared statistically.
In spleens, bacterial loads at the start of treatment were not
statistically significantly different for the three models (P ?
0.060, ANOVA F-test); this was due to the larger standard
deviations in the spleens of the i.v.-infected group. After 1
month of treatment, the bacterial loads in the spleens of i.v.-
infected mice were significantly higher than in either group of
mice infected by the aerosol route (P ? 0.0001, ANOVA
F-test) (Fig. 1B). In addition, the LDA-infected BALB/c mice
showed a lower bacterial count than the C57BL/6 mice (P ?
0.018). After 2 months of treatment (Fig. 1B), drug efficacy in
the spleens was not statistically significantly different for the
three mouse groups evaluated (P ? 0.05, ANOVA F-test). The
spleens of i.v.-infected mice indicated only three mice with
positive cultures (mean, 0.42 log10CFU), whereas the aerosol-
infected mice had sterile cultures. After 4 months of treatment,
mice in all treatment groups failed to show any culturable
bacteria in the spleens.
INH- and MXF-containing combination therapies have sim-
ilar efficacies in the i.v. and aerosol infection models in
BALB/c mice. The purpose of this comparative study was to
evaluate three mouse infection models using BALB/c mice in
one single experiment (after LDA, HDA, or i.v. infection) and
to evaluate three drug regimens in each model. Based on
preliminary experiments (data not shown), a set of parameters
was established (such as starting inoculum and the time be-
tween infection and start of treatment) to ensure similar
starting bacillary burden typical for the HDA and the i.v.
TABLE 1. Bacterial loads in lungs and spleens on the day of
infection and at the start of treatmenta
Expt no. (route)
1 day after
aData are shown for the different experiments performed and illustrated in
the figures, as follows: experiment 1 (Fig. 1), experiment 2 (Fig. 2), and exper-
iment 3 (Fig. 3).
bData are log10CFU counts (or for CFU counts in experiment 1 via LDA
route) in lungs.
cIn experiment 1, both BALB/c and C57BL/6 mice were infected via LDA.
Based on the comparable results and because most laboratories use BALB/c
mice for TB drug evaluations, all further experiments were conducted with the
BALB/c mouse strain.
VOL. 55, 2011MOUSE MODELS FOR TESTING M. TUBERCULOSIS DRUGS1239
models (7 to 8 log10CFU) (38). Treatment included the
standard drug regimen of INH, PZA, and RIF (HRZ), a
MXF-containing regimen where MXF was substituted for
INH in the standard regimen (MRZ), and the combination
of RIF and PZA (RZ). PZA was discontinued in all groups
after 2 months of therapy, except for the dual-drug RIF and
PZA groups. Treatment start for the i.v. animals was at 11 to
12 days, at 14 days for the HDA group, and at day 21 after
low-dose infection. Results are shown in Tables 1 and 2 and
After 2 months of treatment in lungs, similar reductions in
bacterial loads of the three drug regimens were observed for all
three infection models in the BALB/c mice (Fig. 2A, C, and E).
For all three models, the RZ treatment was significantly less
effective than MRZ or HRZ (P ? 0.001), and the efficacies for
both the MRZ and HRZ drug regimens were not significantly
different (HDA, P ? 0.48; i.v., P ? 0.51; LDA, P ? 1.0). Only
in the LDA group did the MRZ treatment group approach
statistical significance for improved activity over the standard
drug regimen at 1 month, but this was statistically not different
from the standard regimen (P ? 0.06). Resistance to RIF of
the remaining colonies was tested after 4 months of RZ treat-
ment in lungs, and all remaining colonies were found to be
susceptible to 4 ?g/ml RIF. Of note, we need to mention here
that the organ homogenates were frozen, and upon thawing for
resistance plating a certain loss of bacterial viability was ob-
served that might have affected our ability to grow all resistant
colonies. After 4 months of treatment, mice treated with either
MRZ or HRZ showed no detectable CFU in lungs. In contrast,
mice treated with RZ showed significantly higher bacterial
loads in lungs (P ? 0.001) and after 4 months of treatment
there were still culturable bacteria in lungs. Based on a Wil-
coxon rank sum comparison, the number of mice carrying
bacteria was significantly higher for the HDA (P ? 0.003) and
i.v.-infected animals (P ? 0.001), but not in the LDA animals
(P ? 0.3). Even after 6 months of therapy with RZ in the HDA
group and the i.v.-infected group, bacteria could be detected.
In spleens a similar trend was seen, although it was less
pronounced than in lungs. After 2 months of treatment, in the
HDA and the i.v. infection models the RZ treatment was again
significantly less effective than the MRZ and HRZ treatment
regimens (P ? 0.0001), and both MRZ and HRZ were as
effective in spleens (P ? 0.36 for HDA and P ? 0.60 for i.v.)
(Fig. 2B, D, and F). In the LDA infection model, after 2
months of treatment, for the HRZ and MRZ treatments the
mice were all culture negative, and six out of seven mice of the
RZ group showed bacterial growth in the spleens. Due to some
culture negativity, the treatment groups were compared using
the Wilcoxon rank sum test (P ? 0.0004). After 4 months of
treatment, the mice from the MRZ and HRZ groups did not
FIG. 1. The i.v. and LDA infection models with M. tuberculosis, using BALB/c and C57BL/6 mice. Data shown are bacterial numbers in lungs
(A) or spleens (B) of BALB/c (f) or C57BL/6 (Œ) mice after an LDA infection with M. tuberculosis or i.v. infection of BALB/c (?) after 4 months
of treatment with INH, PZA, and RIF. Error bars are standard errors of the means.
TABLE 2. Viable M. tuberculosis in lungs and spleens of BALB/c mice 3 months after cessation of treatment with three-drug regimens via
the HDA, LDA, and i.v. infection modelsa
Relapse rate (%)
Log10CFU ? SEM
n/N (%) Log10CFU ? SEM
0.48 ? 0.27
0.75 ? 0.27
0.13 ? 0.13
1.27 ? 0.30
3.14 ? 0.13
1.62 ? 0.35
0 ? 0
0 ? 0
0.53 ? 0.25
0.36 ? 0.25
0.16 ? 0.16
0.13 ? 0.13
0.08 ? 0.08
1.00 ? 0.30
1.70 ? 0.31
0.64 ? 0.26
0 ? 0
0.15 ? 0.15
0.22 ? 0.15
0.10 ? 0.10
aBacterial numbers are presented as the log10CFU with standard errors of the means. Drug treatments included 2 months of MXF, RIF, and PZA followed by 2
months of MXF and RIF (2MRZ/2MR), 2 months of INH, RIF, and PZA followed by 2 to 4 months of INH and RIF (2HRZ/2HR), etc. Relapse rates include all
lung and spleens positive for bacilli. N, total number of mice/group; n, number of mice with detectable CFU.
1240 DE GROOTE ET AL.ANTIMICROB. AGENTS CHEMOTHER.
show any detectable bacterial growth in spleens, and no addi-
tional activity of RZ was seen between 4 and 6 months. At 4
months, mice treated with RZ still had detectable CFU in
spleens in mice of the HDA and i.v. infection model groups,
but no bacterial growth was found in the spleens of mice in the
LDA infection group (P ? 0.0001). After 6 months of therapy
with RZ, the bacterial loads in the spleens from the HDA and
i.v. infection groups remained the same as after 2 or 4 months
of RZ treatment (3.11 and 3.67 log10CFU, respectively). From
the RZ group frozen residual homogenates were plated for
RIF resistance determinance, and only one mouse was found
to have 30 RIF-resistant colonies in the spleen in the i.v.-
infected group. Since the organ homogenates were frozen and
thawed, it is likely that the number of resistant colonies might
have been higher, but we were not able to grow resistant
colonies on agar plates.
Relapse rates were studied for all treatment groups 3
months after cessation of treatment (Table 2). When the re-
lapse rates were compared for the two drug treatments in every
infection model tested, there was no significant difference
within each model. Relapse rates for 2 months of INH, RIF,
and PZA followed by 4 months of INH and RIF (2HRZ/4HR)
were similar to the 2MRZ/2MR groups (HDA, P ? 0.32; i.v.,
P ? 0.75), and for LDA the relapse rate of 2HRZ/2HR was
similar to that of 2MRZ/1MR (P ? 1.0). When the relapse
rates were compared between the tested infection models for
corresponding drug treatment regimens, the i.v. model showed
a significantly higher relapse rate for both the MRZ and HRZ
regimens than for either aerosol model (P ? 0.0001) (Table 2).
Comparison of relapse rates by using Fisher’s exact test for
data from the lungs and spleens revealed that in lungs of
i.v.-infected animals, there were significantly more relapses for
2HRZ/3HR than in the 2MRZ/2MR group (P ? 0.00042). In
the spleens of the i.v.-infected animals, there was again a sig-
FIG. 2. Comparison of HDA and LDA infection models with i.v. infection in BALB/c mice treated with different drug combination regimens.
(A, C, and E) Bacterial numbers in lungs of BALB/c mice after HDA (A), i.v. (C), or LDA (E) infection with M. tuberculosis and 1, 2, 3, 4, 5, or
6 months of drug treatment. (B, D, and F) Bacterial numbers in spleens after HDA (B), i.v. (D), or LDA (F) infection and 1, 2, 3, 4, 5, or 6 months
of drug treatment. Drug treatment regimens included either 2 months of INH, PZA, and RIF followed by INH and RIF (f); 2 months of MXF,
PZA, and RIF followed by MXF and RIF (Œ); or 6 months of PZA and RIF (E).
VOL. 55, 2011MOUSE MODELS FOR TESTING M. TUBERCULOSIS DRUGS1241
nificantly greater relapse seen for 2HRZ/4HR than for 2MRZ/
2MR (P ? 0.04). For the HDA and LDA infection groups, the
2HRZ/3HR treatment groups showed a statistically similar
relapse rate as 2MRZ/2MR for both lungs and spleens (P ?
A different dosing schedule of rifampin in combination with
other drugs does not affect treatment efficacy in BALB/c mice
infected with M. tuberculosis by HDA infection. The goal of the
HDA experiment was to determine the effects of RIF formu-
lation and dosing schedules of drugs in combination treatment
regimens on outcomes of TB infection in BALB/c mice. Two
different RIF formulations with an alternative schedule of drug
delivery were used. In the first method RIF was dissolved in
water and dosed 4 h before other drugs, and in the second
method RIF was administered in 5% DMSO 1 h after combi-
nations of INH or MXF with PZA. PZA was discontinued in
all groups after 2 months of therapy, except for the RZ group.
Infection was initiated in two runs, and mice were randomly
allocated. There were six animals assigned to each treatment
group, and treatment began 14 days post-HDA infection. Re-
sults are shown in Table 1 and Fig. 3. Due to the high mortality
in the RZ arm when we used the second formulation, there
were no mice available for the last time point (3 months after
treatment). We believe the mortality was caused by TB infec-
tion, based on clinical observations. No other treatment groups
had any mortality.
After the first month of treatment in which we compared the
two formulation methods to each other, the bacterial loads in
lungs of mice treated with drugs using method one showed
significantly lower numbers for all treatment regimens com-
pared to mice treated with the method two (across all com-
parisons, P ? 0.001 [two-way ANOVA]) (Fig. 3A and C). The
lung bacterial numbers in treatment groups using formulation
method one were significantly different from each other (Fig.
3A), except for HRZ being similar to RZ (P ? 0.13) and HRZ
being similar to HMRZ as well (P ? 0.48). Lung bacterial
numbers in all treatment groups using formulation method two
were mostly not significantly different from each other (P ?
0.05) (Fig. 3C), except HMRZ was superior to HRZ and RZ.
RIF resistance in frozen homogenates was not detected in RZ
groups by either formulation. After 2 months of treatment,
bacterial numbers in the lungs of all treatment groups were not
significantly different (P ? 0.59, two-way ANOVA). By 3
months, the CFU burdens in the lungs of mice treated with
formulation one were low and statistically similar (P ? 0.05).
At 3 months after treatment using formulation two, there were
no mice left in the parallel RZ treatment group for enumera-
tion of bacilli, and the other treatment arms had reached
After the first month of treatment, the bacterial numbers in
spleens in all treatment groups in the formulation method one
group were similar to those of the method two group for all
treatment regimens (P ? 0.55, two-way ANOVA) (Fig. 3B and
D). For formulation two, MRZ was significantly less effective
than HMRZ (P ? 0.002) or HRZ (P ? 0.007). The bacterial
loads were higher for the RZ group than for any of the other
FIG. 3. HDA infection in BALB/c mice treated with different drug combination regimens, using two different RIF formulations and two dosing
schedules of the drug combinations. (A and B) Bacterial numbers in lungs (A) or spleens (B) of BALB/c mice infected via HDA and treated with
RIF dissolved in water and delivered 4 h before other drugs. (C and D) Bacterial numbers in lungs (C) or spleens (D) of BALB/c mice infected
via HDA and treated with RIF dissolved in 5% DMSO and delivered 1 h after other drugs. The following drug treatments were administered: 2
months of MXF, RIF, and PZA followed by 1 month of MXF and RIF (‚); 2 months of INH, RIF, MXF, and PZA followed by 1 month of INH,
RIF, and MXF (Œ); 2 months of INH, RIF, and PZA followed by 1 month of RIF and INH (f); 3 months of RIF and PZA (E).
1242 DE GROOTE ET AL.ANTIMICROB. AGENTS CHEMOTHER.
drug combination groups. After 2 months of treatment, a sim-
ilar result was observed. HRZ, HMRZ, and MRZ were all
significantly more effective than RZ (P ? 0.001) but were
statistically similar to one another (P ? 0.3). After 3 months of
treatment, for all drug regimens except RZ, the CFU burden
was very low and not significantly different between treatment
groups (P ? 1). The RZ arm with formulation method one
revealed a significantly inferior performance than with any
other arms (P ? 0.001). Due to early mortality, there were no
mice left in the parallel RZ treatment group (formulation
method two) at 3 months for enumeration of bacilli. There was
no RIF resistance detected after plating the lungs and spleens
on RIF-containing plates to account for poor bactericidal ac-
tivity of the RZ regimen; however, drug-free plates at the time
of rifampin plating were not used in this study.
No significant difference was found between pharmacoki-
netic parameters when we used different RIF dosing schedules
in BALB/c mice. Analysis of drug concentrations was per-
formed on plasma samples of mice to assess pharmacokinetics
after single-dose drug exposures to INH, RIF, and PZA, using
the same formulation methods as described above for the ef-
ficacy studies. Pharmacokinetic parameters of INH, PZA, and
RIF were measured after a single dose of RIF was given either
4 h before (in water) or 1 h after (in DMSO) the two compan-
ion drugs, INH and PZA. The results showed that, overall, the
concentrations (in ?g/ml) of all three drugs, INH, RIF, and
PZA, over time were largely similar for both RIF formulation
methods (Fig. 4).
INH concentrations over time were similar when adminis-
tered 4 h after RIF delivered in water or DMSO (Fig. 4A).
When INH was administered 4 h before RIF, the INH levels
were, as expected, very low at the 4-h time point, due to the
short half-life of INH (Fig. 4B). The low INH drug levels did
not allow us to make direct statistical comparisons between the
two formulations. INH drug concentrations were similar
whether the drug was administered as a single agent or com-
FIG. 4. Single-dose pharmacokinetic plasma drug levels of standard TB drugs in BALB/c mice when the drugs were administered as drug
combinations. (A) INH drug levels over time when administered with PZA 4 h after RIF in DMSO (}) or water (?). (B) INH concentrations when
administered with PZA 4 h before RIF was given in DMSO (}) or water (?). The data in the graphs are shown from the time of RIF
administration. (C) INH concentrations when INH was given alone (}) or in combination with PZA (?) without RIF. (D) RIF concentrations
after PZA and INH, RIF in DMSO (}), or water (?). (E) RIF concentrations when RIF in DMSO (}) or water (?) was given 4 h before PZA
and INH. (F) RIF plasma levels when given alone in water (?) or DMSO (}). (G) PZA concentrations when RIF was administered 4 h before
PZA and INH, with RIF dissolved in DMSO (}) or water (?). (H) PZA concentrations when PZA and INH were given 4 h prior to RIF dissolved
in DMSO (}) or water (?). (I) PZA plasma concentrations when PZA was given alone (}) or in combination with INH (?). Doses: INH, 25
mg/kg; RIF, 10 mg/kg; PZA, 150 mg/kg (as a single dose by oral gavage). All time points refer to the time after RIF administration.
VOL. 55, 2011MOUSE MODELS FOR TESTING M. TUBERCULOSIS DRUGS 1243
bined with PZA in water (Fig. 4C). PZA groups demonstrated
similar drug concentrations over time when administered after
RIF (Fig. 4G) when RIF was administered in either water or
DMSO. When PZA was administered 4 h before RIF, the
plasma levels of PZA were, as expected, very low at the 4-h
time point, due to the short half-life of PZA (Fig. 4H). The low
PZA drug levels did not allow us to make direct statistical
comparisons between the two formulations. PZA levels did not
differ over time when administered alone or combined with
INH (Fig. 4I).
The median AUC0–?values for single drugs were deter-
mined in order to compare the drug combinations when ad-
ministered in water versus DMSO. The AUC0–?for RIF was
slightly higher in water versus DMSO, 97.46 and 86.02 ?g ? h/
ml, respectively (?13%). After a single dose of INH adminis-
tered with RIF in water compared to RIF in DMSO, the
AUC0–?of INH was 24.41 and 19.34 ?g ? h/ml, respectively
(?26%). Lastly, the AUC of PZA was 433.95 and 419.38
?g ? h/ml when PZA was given with RIF in water versus
DMSO, respectively (?3%). When administered as a single
agent, RIF demonstrated a median Cmax_D(the concentration
maximum, normalized to dose) of 47.83 and 60.83 ?g/ml in
water versus DMSO, respectively (?21%). When RIF was
administered in combination with INH and PZA, the Cmax_D
of RIF was 39.14 and 32.93 ?g/ml in water versus DMSO,
In preclinical testing of experimental compounds against
tuberculosis, various mouse models have been used by differ-
ent investigators in the field. This study is the first, to our
knowledge, to evaluate the most widely used mouse infection
models side by side in one laboratory. The rationale of this
work was to understand the most crucial parameters of the
mouse infection models that may change the outcomes of drug
efficacy trials in TB drug development.
Different drug combination treatment regimens were evalu-
ated in three different mouse infection models: the LDA,
HDA, and i.v. infection mouse models, using BALB/c mice. An
initial experiment indicated that the treatment efficacy of the
standard drug regimen, HRZ administered over 4 months, was
equivalent for BALB/c and C57BL/6 mice infected via LDA.
Since most laboratories use BALB/c mice for TB drug evalu-
ations, we opted for this mouse strain for all further compar-
ative studies. The experiments aimed to achieve the same bac-
terial loads in the lungs at the start of treatment and mimic the
protocols used by other investigators in the field employing
HDA or i.v. infection models. The results of the mouse studies
showed that the killing kinetics of the drug regimens in lungs
were significantly slower for the i.v.-infected versus aerosol-
infected animals in lungs as well as in spleens. The reason for
this is not entirely clear but might be explained by the state of
the bacilli (the proportion of intra- versus extracellular bacilli)
as well as the growth rates of the bacilli being possibly slower
in the i.v. model. In addition, the i.v. route of infection induces
an altered state of lung immunity, compared to the direct
deposition of the bacteria in the lungs by the other routes (19),
which might alter the responsiveness of the bacteria to treat-
The efficacy results of the three infection models showed
that the MXF-containing regimen (MRZ/MR) reduced the
bacterial load in a statistically similar way as the standard
regimen (HRZ/HR), until no detectable bacilli could be de-
termined in lungs and in spleens. Therefore, all three models
showed a similar outcome based on drug combination com-
parisons regardless of the mouse model used. In all three
models, the RZ combination was far less efficacious than any of
the three-drug combinations. Drug resistance was likely not an
issue, as no RIF-resistant colonies were obtained after plating
on 4 ?g/ml of RIF, although isolates having a lower level of
resistance could have been missed. The killing kinetics for RZ
also differed between lungs and spleens. In the lungs a steady
reduction in bacterial numbers was generally seen in the aero-
sol infection models, while in spleens after 1 month of treat-
ment the RZ activity was significantly reduced in the HDA
model. Whether this was caused by a difference in replication
rates of the organism in the different organs, the distribution of
the bacteria in the host, or lower drug penetration into the
different organs is not clear at this time.
Another measure of treatment efficacy, namely, relapse of
infection rate, was evaluated 3 months after cessation of drug
treatment in the different mouse infection models. The 2MRZ/
2MR-treated group showed statistically similar relapse rates as
the 2HRZ/4HR group in the HDA and i.v. infection models. In
fact, the relapse studies revealed that for the i.v. infection
model, the standard drug regimen (HRZ) showed a signifi-
cantly higher number of mice relapsing after 5 months of
treatment than the MXF-containing regimen (MRZ) after 4
months of treatment. This result shows the superior sterilizing
activity of the moxifloxacin-containing regimen over the stan-
dard regimen in the i.v. infection model. In the HDA model, a
similar trend was observed between the 5-month standard reg-
imen and the 4-month treatment with the moxifloxacin-con-
taining regimen, although this result was not statistically sig-
nificant. In the LDA model, virtually no relapse was observed
after 2MRZ/1MR treatment, while there was a 10% relapse
seen after the 2HRZ/2HR treatment. Although the relapse
rates for the mice in the LDA group were very low, resulting in
an insufficient statistical power to allow a statement of the
significance, this was a meaningful result, as it showed a trend.
We published previously the statistical limitations of relapse
studies, pointing out that small studies can still be useful as
“screening” experiments (25). In that earlier study, we calcu-
lated the need for obtaining a difference in relapse rates of a
?40 to 50% difference between the comparison groups in
order to achieve a power of 0.8 when using 20 mice per group
(25). In these studies only a difference of 40 to 50% in relapse
rates was observed for the i.v. infection group, and therefore a
strong statistical statement can be made only for this infection
group, whereas for the aerosol infection groups only a trend of
relapse rates can be shown. Nevertheless, for all three infection
models in BALB/c mice, a similar trend was seen regarding the
relapse of infection in the MXF-containing regimen versus the
standard drug regimen, regardless of the mouse infection
In contrast to observing similar efficacies of MRZ and HRZ
over the initial months of treatment as described above, the
relapse rates for the MXF-containing regimen (2MRZ/2MR)
were significantly lower for the i.v. infection group than for the
1244 DE GROOTE ET AL.ANTIMICROB. AGENTS CHEMOTHER.
standard drug regimen (2HRZ/3HR). And, in the aerosol in-
fection models, a similar trend was observed. These results
show the importance of such relapse studies, versus only eval-
uating the bactericidal efficacies of drugs in mouse models
during treatment in order to select the drug regimen that can
lead to a cure. That bactericidal activity during treatment does
not always provide a good correlation with relapse data has
been seen before in a mouse study at John Hopkins University
(38). Of importance, a recent paper by Koen Andries et al.
demonstrated that bactericidal potencies of new TB drug reg-
imens do not always predict relapse potential (2). In that study,
the investigators rank ordered the bactericidal and sterilizing
potencies of several regimens and found that drug regimens
with very good bactericidal properties did not necessarily have
good sterilizing properties. For instance, treatment with a
three-drug regimen (TMC207, PZA, and MXF) for 4 weeks
resulted in culture conversion, but 5 months of treatment with
the same regimen was needed to achieve acceptable relapse
rates. In contrast, treatment with the regimen of TMC207,
PZA, and rifapentine (RPT) for 4 weeks did not result in
complete culture conversion, but only 3 months of treatment
was needed to achieve an acceptable relapse rate. In these
studies, it was clear that MXF was more bactericidal than RPT
but that RPT was far more sterilizing (2). The results pre-
sented provided additional credence to the inclusion of relapse
studies as part of the preclinical evaluation in order to assess
the true sterilizing potential of a new regimen. Ultimately, the
clinical trials conducted with these regimens will provide def-
inite answers to the question of the predictive value of bacte-
ricidal versus sterilizing activities seen in animal models, and
they are required for further validation of the animal model
The i.v. infection model showed a significantly greater rate
of relapse than either aerosol infection model. After an i.v.
infection, bacteria are primarily retained in the liver (90%) and
spleen (10%), and only 1% will implant into the lungs (36, 43).
We found in our first experiment, when we evaluated the two
mouse strains, most bacilli were found in the livers (99%),
compared to 0.3% in lungs and 0.4% in the spleens. Therefore,
the difference in relapse could be caused by a difference in
immune response generated between the i.v. and aerosol in-
fection models from the start of infection. Host immune re-
sponses, such as innate, adaptive, and memory immune re-
sponses, including T regulatory cell populations, have recently
been found to account for differences in outcomes of animal
TB infection studies and could be at play in the different
models (19, 42).
When comparing our data with earlier published data, our
i.v. infection groups showed a higher rate of relapse in the
HRZ group than results reported in a recently published study
(55% versus 17%, respectively) (21), while for the HDA-in-
fected groups relapse was very similar to studies described in
the literature (5% versus 0%, respectively) (41). For the i.v.
infection models, our studies also revealed higher relapse rates
than in historical trials that were performed in Paris at either
Institut Pasteur in the 1950s, 1960s, and 1970s (13–15), in the
Pitie ´-Salpe ˆtrie `re School of Medicine from the 1980s to the
present (11, 24), or those performed at Cornell University
(30–33). These important historic studies, which reflect the
human clinical relapse rate, often used outbred Swiss mice,
which may have resulted in different outcomes and could ex-
plain these results. A more likely explanation is that our studies
may have resulted in a higher CFU burden at the start of
therapy. In general, aerosol infection models are more expen-
sive, due to the necessity to purchase and maintain a special-
ized aerosol apparatus; however, this model may be more
relevant to human infection with TB, which is acquired by
direct implantation into the alveoli. The human infectious in-
oculum is more closely recapitulated by the LDA rather than
the HDA model. The LDA model never achieves a bacterial
load greater than 6 to 6.5 log10CFU in immunocompetent
animals and therefore has a smaller window in which one can
assess bactericidal activity compared to the HDA and the i.v.
models, and thus the treatment periods chosen should be care-
fully assessed and shortened. On the other hand, the HDA
model achieves a bacterial burden of 7 to 8 log10CFU, thereby
simulating a human cavitary lesion. However, in humans there
are usually only one or a few cavitary lesions, while in a mouse
with an equivalently high burden, there will be hundreds of
lesions. Due to the high bacterial burden at the start of treat-
ment in the HDA model, it is possible to determine the resis-
tance frequency of a drug or drug combination.
In the studies presented here, the combination of HRZ was
in all three infection mouse models significantly more effective
than RZ dual therapy. In none of the three infection models
was an antagonism of H in the HRZ combination observed.
Antagonism between the three standard drugs has been shown
by others (10), where it was demonstrated that the dual regi-
men of RZ after removal of INH performed better than the
standard three-drug regimen HRZ. However, this antagonism
was not always seen to the same extent by the same investiga-
tors, and the antagonism was recently suggested to be depen-
dent on the INH concentration (especially at 25 mg/kg) (1). In
the studies described here as well as in studies reported by
others, INH is administered at 25 mg/kg, and in our case
antagonism was never observed. In one other historic study in
mice, PZA actually antagonized the bactericidal effect of INH
when treatment with both agents was started within 20 min of
infection, and this effect diminished entirely when treatment of
an established infection was evaluated (30). Certain conditions
appear to influence standard TB drug therapy, such as sequen-
tial administration of INH followed by PZA, showing different
efficacies than when INH and PZA are simultaneously admin-
istered (34). In the studies presented here, we also found some
variations between experiments, with HRZ being far more
effective than RZ in the initial experiment, while this difference
was less pronounced in a second experiment. However, true
antagonism between the standard drugs was never observed,
and HRZ always showed equivalent or better activity than RZ.
Similar to our results, other investigators using models of in
vitro (23) and in vivo combination drug efficacy experiments
have failed to find antagonism (9). Others have also described
better or equivalent activity of HRZ versus RZ (20). In the
reports by Ibrahim et al., no antagonism was found; in fact,
HRZ was found to decrease the bacterial burden in lungs by
1.5 log10CFU greater than RZ in their model after 1 month of
treatment (20). In addition, results in their laboratory showed
the combination of MRZ to be as effective as HRZ and equiv-
alent to RZ at 2 months of treatment. The same authors also
found that an MRZ regimen for 4 months resulted in a relapse
VOL. 55, 2011 MOUSE MODELS FOR TESTING M. TUBERCULOSIS DRUGS 1245
rate that was not significantly different from that observed after
the HRZ regimen for 6 months (21). Their mouse model uses
the M. tuberculosis H37Rv strain, Swiss mice, and a high-dose
i.v. infection model. Grosset, Nuermberger, and colleagues
observed antagonism in HRZ that resulted in less activity than
RZ with the M. tuberculosis H37Rv strain, BALB/c mice, a
high-dose aerosol infection model, and dosing of RIF at least
1 h ahead of HZ (12, 39). As highlighted by Nuermberger in a
recent publication, until the specific conditions of antagonism
of standard drugs in certain laboratories are fully delineated, it
is recommended that future studies of drug combinations in-
clude appropriate control arms in long-term trials (37). We
absolutely agree with this statement and strongly urge investi-
gators new to the field that they should, for every study that
substitutes a single agent in the standard HRZ regimen, in-
clude both an HRZ and an RZ treatment arm in their mouse
The significance of the antagonism between the drugs in the
HRZ regimen is most apparent when comparing a new drug
regimen to the standard drug regimen prior to conducting
clinical trials. With an observed antagonism between the three
drugs of the standard regimen, one might quickly interpret
data for a new drug regimen as being superior to standard
therapy. Recent mouse trials with MXF substituted for INH
showed significant improvement in efficacy over standard ther-
apy (HRZ), and most of this benefit was attributed to removal
of the antagonism between INH with RIF plus PZA (37, 40,
41). The results of our mouse studies showed a superior activ-
ity with the substitution of MXF for INH in the standard
regimen, based on the relapse studies, while no advantage was
seen for the bactericidal activity during treatment. The results
of our mouse studies support the findings of human TBTC
Study 28, in which no significant difference was seen in bacte-
ricidal activity when MXF was substituted for INH in a daily
regimen for 8 weeks (11). This leads to the question of whether
the 8-week sputum conversion results in patients will be pre-
dictive for the sterilizing activity of a regimen measured by
relapse after 2 years. Sputum samples contain only one com-
partment of the bacterial population, whereas the bacteria in
the cavitary lesions within the lungs might have a completely
different phenotype and environment, which will affect their
drug responsiveness. The mouse studies predict that MXF
might only show significant benefit in later stages of a clinical
trial. The long-time follow-up in the ReMox TB clinical trial
might bring the definite answer to these questions, and this
trial is under way (www.tballiance.org).
In the past, the dosing schedule of RIF and formulations of
drugs in the standard drug regimen for tuberculosis have been
described to have a significant effect on the pharmacokinetics
in mice (12). In the studies described here, we evaluated the
pharmacokinetics of the individual drugs of the standard reg-
imen in different dosing schedules and formulations of RIF
when combined with INH and PZA. Besides the pharmacoki-
netics, we also studied the effects of RIF formulation and
dosing schedule on the efficacy of the drug combination by
using the HDA model in BALB/c mice. The different formu-
lations and dosing schedule of RIF with other drugs in com-
bination gave similar efficacy results in lungs and spleens. In-
terestingly, in the mice treated with RZ using formulation
method two, a higher mortality was observed than in mice
treated with RZ in formulation one. The combination of RZ
has been shown to have significant adverse effects in mice (29)
and has led to mortality in TB patients (3). The reason for the
mortality with only one formulation in our studies reported
here is unclear at this time. There were some differences seen
in the kinetics of the concentration-time curves of RIF that
depended on whether the drug was ground in a mortar in water
or prepared as a DMSO solution, but overall drug exposures of
RIF were not different. In this study, we did not address the
situation when RIF was given simultaneously with INH and
PZA, as this was beyond the scope of our studies.
One last variable not studied in this work is the bacterial
strain used for the infection of mice. With the results obtained
here, the study of different bacterial strains (laboratory and
clinical M. tuberculosis strains) is the current focus of our
laboratory for extensive in vitro and in vivo drug combination
evaluations. We also realize that the studies described here
only evaluated the standard drug combination and MXF-con-
taining drug combinations, and therefore more combination
regimens should be tested in the future in the different infec-
tion models. In summary, our studies demonstrate that the
evaluation of TB drug regimens in mouse M. tuberculosis in-
fection models do not differ between the two mouse strains, by
the inoculum size or the route of infection used, for drug
efficacy as well as for relapse of infection. In addition, the
organ CFU counts of the standard drug regimen were not
affected by a difference in formulation or dosing schedule of
RIF in combination with INH and PZA. Significant differences
in results of long-term efficacy mouse studies are, however,
observed between various laboratories, such as is seen with the
phenomenon of antagonism between the standard drugs INH,
RIF, and PZA. Given that many variables are present in ani-
mal studies for drug development, we therefore recommend
that preclinical animal studies not be standardized but that
critical studies be confirmed in a second laboratory using a
different strain of M. tuberculosis and a different animal model,
and that such studies include the appropriate treatment con-
trol groups, prior to moving to expensive human trials.
We thank Phillip Chapman (Statistics Department, Colorado State
University) for statistical assistance, Jena Valdez, Courtney Hastings,
Amanda Marsh, and Jordan Clark for technical assistance, and the
Laboratory Animal Resources staff for assistance with care and anal-
ysis of the animals. We give a special thanks to the Centers for Disease
Control and Prevention Tuberculosis Laboratory for sequencing the
pncA gene product and to Leonid Heifets and the staff of the Myco-
bacteriology Laboratory at the National Jewish Health, Denver, CO,
for the MIC testing of our M. tuberculosis strain.
This project was supported by the Bill and Melinda Gates Founda-
tion under Drug Accelerator grant ID number 42589, “Assay Stan-
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